1. Field of the Invention
The present invention relates to an immersion microscope objective, and a microscope using the same.
2. Description of the Related Art
In the field of research of biology, suppressing invasive potential toward living cells to be as low as possible, and being capable to observe an extremely minute structure (hereinafter, ‘microscopic structure’) of a specimen, have always been sought. In recent years, for observing the microscopic structure of a specimen, there have been an increasing number of applications for observing a specimen by using a weak light. Light diffracted from the microscopic structure is a light having a large angle of diffraction. When an image is formed by using diffracted light having a large angle of diffraction, it is possible to observe the microscopic structure.
One of the applications for observing a specimen by using weak light is a total internal reflection fluorescence observation. In the total internal reflection fluorescence observation, evanescent light is used. Evanescent light is generated by making incident illumination light on a boundary surface having different refractive index, at an angle of incidence not smaller than certain degrees, and by making the illumination light undergo total internal reflection. The evanescent light has a peculiarity of being localized in an area smaller than a wavelength on an opposite side of the illumination light with respect to the boundary surface, and not propagating in a free space.
In the total internal reflection fluorescence observation, light is made to be incident on a cover glass such that the total internal reflection occurs at a boundary surface of the cover glass and a specimen. As the light is incident on the cover glass and undergoes total internal reflection, evanescent light is permeated from the boundary surface toward the specimen. An area through which the evanescent light is permeated is an area of about a wavelength of light. In such manner, an area illuminated by excitation light is restricted to the area through which the evanescent light is permeated, or the area of about the wavelength of light. In the total internal reflection fluorescence observation, fluorescence is not generated in an area except the limited illuminated area. Therefore, in the total internal reflection fluorescence observation, fluorescence observation with less background noise is possible.
Furthermore, the evanescent light has a peculiarity that a depth of an area through which the evanescent light is permeated changes according to an angle (an angle of incidence) of light which is incident on the boundary surface. Larger the angle of incidence of light, narrower is the area through which the evanescent light is permeated. When the angle of incidence of light is made large, since it is possible to reduce the background noise, even brighter observation with a high resolution is possible. The total internal reflection fluorescence observation having such characteristics has been used for observing a movement and an activity of various substances in a biological cell.
In the total internal reflection fluorescence observation, a microscope objective with a large numerical aperture is used. For instance, when the specimen is a cell, a refractive index of a cell is in a range of 1.33 to 1.4. Therefore, for letting the total internal reflection occur at the boundary surface of the cover glass and the specimen, the numerical aperture of the microscope objective is required to be at least 1.4 or larger than 1.4. Moreover, for narrowing the area through which the evanescent light is permeated, it is necessary to make large the angle of incidence of light which is incident on the boundary surface. For fulfilling the abovementioned requirements, it is desirable that the numerical aperture of the microscope objective is large.
Moreover, even in an observation other than the total internal reflection fluorescence observation, when it is possible to take in diffracted light with a large angle of diffraction, it is possible to observe the microscopic structure. Therefore, it is desirable that the numerical aperture of the microscope objective is large.
As a microscope objective with a large numerical aperture, immersion microscope objectives disclosed in Japanese Patent Application Laid-open Publication Nos. 2002-098903, 2002-148519, and 2006-113486 are available.